Protein phosphorylation, one of the most common and important post-translational modifications, plays a pivotal role in the control of many biological processes such as cell growth, division, and signaling. Dysregulation of the phosphorylation-mediated signaling pathways has been linked to many diseases such as cancers and heart diseases. Therefore, the effective capture, separation, and comprehensive analysis of phosphoproteins from complex biological samples are crucial for understanding fundamental cell biology and disease mechanisms as well as disease diagnosis, but remain a major challenge. We herein aim to solve the challenge towards a comprehensive analysis of the phosphoproteome by developing a class of smart multivalent nanoparticles (NPs) for enriching phosphoproteins globally out of complex biological samples followed by top-down mass spectrometric (MS) analysis of intact phosphoproteins. This interdisciplinary approach integrates the exciting innovations in both nanotechnology and top-down mass spectrometry-based proteomics and capitalizes on their complementary strengths. It has significant advantages over the conventional methods for phosphoproteomics including: high capturing capacity due to high surface area of NPs, high binding affinity due to antibody-like multivalent characteristics of NPs, highly specific and effective capture of phosphoproteins, universal enrichment of all types of phosphoproteins unlike antibody approach, preservation of phosphoprotein activity for protein functional studies, and comprehensive characterization of phosphoproteome and mapping all phosphorylation sites with 100% sequence coverage. We will synthesize the multivalent NPs using magnetic or non-magnetic materials and functionalize them with metal chelate ligands that can selectively and reversibly bind to phosphate groups. We have demonstrated the proof-of-principle of this technology and showed the enrichment is highly specific and effective using model protein mixtures and the NPs we have synthesized. We will continue to improve the performance of this enrichment method using model protein mixtures and proteins from heart tissues. The captured intact phosphoproteins will be further separated by multidimensional liquid chromatography and analyzed by high resolution top-down mass spectrometry we have developed or are developing. This innovative protein-based technology for effective separation and analysis of intact phosphoproteins will potentially transform the paradigm of the main-stream phosphoproteomics. The success of this research will provide a powerful new technology to researchers in the entire biological and basic medical research communities, particularly proteomic researchers, enzymologists, and cell biologists. PUBLIC HEALTH RELEVANCE: This proposal seeks to develop novel smart nanomaterials for highly specific and efficient capturing of phosphoproteins globally out of complex proteome followed by comprehensive proteomic analysis of the captured whole phosphoproteins. The success of this proposal will have significant impacts on understanding molecular mechanisms of many diseases by delineating phosphorylation-mediated cell signaling pathways, and could enable the development clinical diagnosis assays for diseases where phosphoproteins serves as biomarkers.